Browsing by Author "McDonnell, Donald P"

The steroid hormone progesterone has complex physiologic effects. In typical development and function, cells respond to progesterone in a dose- and tissue-specific manner. Despite the wide range of physiologic concentrations, canonical effects of progesterone have been characterized in the context of a high physiologic dose (10nM+), relevant during uterine cycling. This narrow focus has produced a gap in knowledge, particularly as it relates to the effects of post-menopausal low concentration progestins (0.1-0.3nM). Given that healthy tissues possess regulatory mechanisms to sense and respond to progesterone in a non-linear dose-specific manner, we hypothesized that breast malignancies would also display discontinuous dose-specific dynamic responses. Our results show that treatment with low dose progestins (0.1-0.3nM) drives proliferation in T47D human breast cancer cells, while high dose progestins (10nM+) inhibit proliferation. Using both unbiased and targeted approaches, we found that low dose progestins facilitate cell cycle entry by enhanced expression of CCND1 (cyclin D1) and SGK1 (serum and glucocorticoid related kinase 1), which are required for initiation of the downstream molecular cascade including phosphorylation of retinoblastoma protein (Rb) and expression E2F1. Expression of CCND1 and SGK1 mRNA are proximal responses to low dose progestin treatment, but transcriptional activation is not mediated by canonical progesterone receptor (PR) activity. Future work is needed to identify previously unexplored mechanisms of PR action in the context of low dose progestin treatments. In summary, these results challenge the assumption of dose response linearity to progestins and show unique functional and molecular effects of low dose progestin treatment. Of potential concern, our findings suggest that certain breast cancers, especially those expressing high levels of PR, may be accelerated by normal post-menopausal circulating concentrations of progestins (0.1-0.3nM). However, these findings also offer a sound rationale for the clinical therapeutic use of high dose progestins for patients with PR+ breast cancer.

Every cell in our body contains the same genetic material, but what differentiates one cell type from another is the way in which that material is interpreted. Hidden in the 98% of non-coding DNA sequences, once referred to as “junk DNA,” are the instructions for how to turn genes on and off (i.e., the operating system). As in any other language, decoding the instructions between DNA and gene expression is the key for understanding transcriptional regulation. Without understanding the grammar of transcriptional regulation, we cannot tell which sequence changes affect gene expression and how.

In this study, we have focused on defining the mechanisms mediating gene expression in response to steroid hormones (predominantly 17β-estradiol) as a model system for other non-steroid transcription systems that can be exploited to define general principals governing steroid responsiveness upon target genes. In particular, we have demonstrated that DNA sequence constraints define the functionally active steroid nuclear receptor (sNR) gene regulatory elements in the genome, and this functionality is restricted to elements that vary from the consensus palindromic elements by one or two nucleotides, named nuclear receptor functional enhancers (NRFEs). At NRFEs, the chromatin binding of steroid nuclear receptors is not only correlated with active eRNA production, but also RNAPII occupancy as well as hormone-dependent coregulator and transcription factor (TF) recruitment. Moreover, steroid nuclear receptors with mutated DNA binding domains (DBD), were shown to still interact with chromatin, yet lack hormone-dependent transcriptional activity, highlighting the fact that steroid nuclear receptors can interact with chromatin in a transcriptionally inactive state (i.e., the majority of sNR chromatin interacting events identified in ChIP studies are not linked directly to transcriptional events) (Chapter 2). We further demonstrate that the palindromic architecture of the regulatory element is the underlying mechanism that governs chromatin interaction by steroid nuclear receptors, and the non-functional chromatin interacting sites (non-NRFEs) observed in ChIP-seq studies are subject to the same rules and constraints as NRFEs. Thus, NRFE vs. non-NRFE binding is dictated at the individual nucleotide level, and the residence time (or strength of binding) is determined by the number and locations of variants within the consensus element. The basis of these rules and DNA constraints follow specific algebraic relationships. These findings quantitatively define how steroid nuclear receptors select the ‘appropriate’ regulatory targets out of a very large number of highly similar sequences in the genome, thus eliciting a specific cellular response (Chapter 3).

Estradiol is a potent mitogen in the mouse uterus, a well-characterized tissue used to study the underlying mechanisms of estrogen-mediated transcriptional regulation. Previously shown, estradiol-mediated transcriptional regulation in the mouse uterus is biphasic and can be divided into initial (early) phase and subsequent (late) phase transcriptional events. In this study, we demonstrate that late phase estradiol-mediated transcription requires the early phase transcripts and low-affinity estrogen receptor α (ERα) ligands cannot sustain late phase hormone-mediated transcriptional events. In addition, the interaction of ERα with chromatin is (1) immediate, (2) does not change locations after initial contact, (3) is retained the longest at NRFEs, and (4) is depleted prior to the late phase transcriptional events. Collectively, this indicates that estradiol-mediated late phase transcripts are regulated secondary to early induced transcripts (i.e., the early induced transcripts activate other transcription factors which are responsible for producing the late phase transcripts). Furthermore, the AF2 coactivator surface of ERα is not required for hormone-dependent ERα recruitment to NRFEs, estrogen-independent basal transcription requires ERα binding at NRFEs and the growth factor insulin-like growth factor 1 (IGF-1) activates ERα by recruitment of ERα to NRFEs (Chapter 4).

Our understanding of the physiology and transcriptional regulation of steroid hormones was significantly advanced following the generation of mutant mouse models possessing disruptions (knockouts) of the steroid nuclear receptor genes. In this study, we identified the molecular defects caused by a homozygous missense mutation in ERα identified in an 18 year-old woman with complete estrogen insensitivity syndrome; a clinical presentation similar to those of ERα knockout (αERKO) female mice. From these studies, we identified a potential therapeutic, Diethylstilbestrol (DES), for treating this estrogen insensitivity condition. Treatment of this patient with DES is underway and we remain involved as collaborators in this clinical study. Our studies also characterize the molecular defects caused by a different homozygous mutation in ERα identified in two sisters and a brother that likewise exhibit complete estrogen insensitivity (Chapter 5).

Hormone-dependent transcriptional regulation requires the recruitment of coregulators to the regulatory regions of target genes. This recruitment is determined by the overall surface topography of the sNR. Phage display is a widely-used research technique for screening highly diverse peptide libraries to enrich for sNR-binding clones. It requires two primary components for affinity selection: (1) a phage display cDNA library, and (2) purified recombinant sNR protein. High level expression of soluble biologically-active sNR protein is particularly challenging due to its largely hydrophobic ligand binding domain. In this study, we overcame this challenge by constructing a protein expression system that provides the factors responsible for protein folding of sNRs (Hsp90, Hsp40, Hsp70, Hop and p23) at levels comparable with the amount of over expressed sNR (Chapter 6). Affinity selection in phage display involves panning of a phage library to enrich for sNR-binding clones followed by their amplification. This amplification step enriches for clones that have a growth advantage, introducing bias into the selection that favors faster growing clones regardless of the selection pressure. To eliminate this bias, individual phage must be separated into different growth chambers so they cannot compete for bacterial hosts. To do this, we used microfluidic flow-focusing technology (MFFT) to generate monodisperse droplet based compartments to encapsulate individual phage clones and achieve non-competitive amplification of millions of phage clones having different growth characteristics. The elimination of growth-based competition ensures that selection of binding clones is driven only by the binding strength of each clone for the sNR. The successful development of a MFFT platform and proof of principal demonstration, allowed us to then implement a high throughput MFFT droplet system. This project was the first application to introduce this novel technology to the National Institute of Environmental Health Sciences (NIEHS) (Chapter 6).

Transcriptional regulation takes place at the level of single cells. However, many traditional techniques involve homogenizing tissue samples composed of millions of cells, and thus can only deal with population averages. Single-cell sequencing reveals the inherent properties of a single cell from the large scale of the genome, information critical for understanding cellular heterogeneity in cancer and response/resistance to therapy. Using our new high throughput MFFT droplet system, genome-wide gene expression profiling of individual cells can be done by separating thousands of individual cells into nanoliter-sized aqueous droplets, associating a different barcode with each cell’s RNAs, and sequencing them all together. This results in transcripts from thousands of individual cells that are all identified by their cell of origin. Here, we establish a droplet microfluidic method to sequence genomes of single cells from dissociated, complex tissues using a custom fluorosurfactant (i.e., a triblock copolymer consisting of a polyethylene glycol (PEG) center block covalently bound to two perfluorinated polyether (PFPE) blocks by amide linking groups), to address two of the major challenges in performing biological, drop-based assays: to stabilize aqueous droplets in fluorocarbon oils and to make the droplets compatible with biological molecules and cells (Chapter 6).

Post-translational modification by SUMO is an important mechanism to regulate transcription. Tamoxifen is used in the treatment and prevention of ER positive breast cancer. Tamoxifen is metabolized predominantly by the cytochrome P450 system to several primary and secondary metabolites, some of which exhibit more antiestrogenic effects than tamoxifen itself. In this study, we demonstrate that the more antiestrogenic effect of endoxifen versus tamoxifen is due post-translational modification by SUMO and inhibition of SUMO derepresses endoxifen’s anti-estrogenic activity. This mechanism of transcriptional repression was also demonstrated in other antiestrogens including fulvestrant, raloxifene, bazedoxifene, idoxifene and lasofoxifene (Chapter 7).

Breast cancer is the most diagnosed cancer and the leading cause of cancer-related deaths among women worldwide. Estrogens, acting through their cognate receptor(s) support the growth of most breast cancers and as such, endocrine therapies that target cancer cell intrinsic signaling by estrogens are the cornerstone of treatment for these patients. While these therapies have increased the survival of patients, resistance remains an impediment to durable clinical responses especially in patients with metastatic disease. Thus, there is an unmet medical need for new therapies to treat this disease. Recently, therapies to increase antitumor immunity have led to clinical success in various cancers, but their efficacy in ER-positive (ER+) breast cancers is still limited. Interestingly, studies outside the realm of cancer have established that estrogen signaling affects immunological responses. However, how estrogen signaling in ER-positive tumor infiltrating cells affects cancer progression and metastasis is underexplored. Consequently, the overarching goal of this thesis work was to decipher how estrogen signaling and endocrine therapy in the tumor immune microenvironment affect tumor progression. This work was undertaken with the goal of identifying processes and pathways, the targeting of which could improve antitumor immunity and increase the efficacy of endocrine therapies.

The first part of this work will describe our efforts to define the role of estrogens/estrogen signaling on the function of natural killer cells in breast cancer metastasis. In these studies, we utilized murine breast cancer cell lines that are themselves insensitive to estrogens and investigated how the most common estrogen, 17β-estradiol (E2) affected breast cancer metastasis of these cells in immunocompromised mice and when propagated syngeneically in immunocompetent mice. We report that much of the biology of estrogens in tumors/metastatic lesions can be attributed to their actions in immune cells. Notable was the observation that E2 decreases metastasis of breast cancer cells in immunocompromised mice, but these protective effects are lost or reversed in immunocompetent mice. Immune profiling of the estrogen-treated mice indicated that E2 reduced the numbers, activity of NK-cells and their ability to produce IFN. These activities reduced their antitumor potential. Using single-cell RNA-sequencing technology (scRNA-seq), we identified an immunosuppressive sub-population of NK cells characterized by high expression of transforming growth factor B (TGFβ) in metastasis-bearing lungs that is expanded upon estrogen treatment in mice. Additionally, we discovered that E2 inhibits NF-B signaling and suppresses the activities of transcription factors involved in IFN production. Using a mouse model of NK-specific ER-alpha (ER) ablation, we confirmed that the actions of estrogens in NK cell biology were mediated through direct actions of the receptor in these cells. We also report unexpected pharmacology of an ER degrader, fulvestrant in breast cancer metastasis in that, like E2, fulvestrant inhibited NK cell numbers and activity and increased breast cancer metastasis. Collectively, our data indicated that estrogen signaling in NK cells suppresses the activity of these immune cells and contributes to increased breast cancer metastasis. These findings beg a reevaluation of mechanisms by which ER-modulators/endocrine therapies impact NK cell function, work that we believe will enable the selection from among existing drugs those which have favorable activities in NK cells and/or inform approaches to develop the next generation of ER-modulators optimized for their activity in metastatic disease.

While most of this work focuses on estrogen signaling in NK cells, the second part of this thesis describes our studies investigating how estrogens affect the function of tumor-associated macrophages (TAMs) in the context melanoma primary tumor growth. Melanoma is not traditionally classified as a hormone-sensitive cancer, however, there exists a gender bias in response to immunotherapies in melanoma, with women experiencing worse outcomes than men. Our previous data indicated that estrogens increase the growth of primary melanoma tumors, decreasing the number of tumor-infiltrating T-cells, and skewing TAMs towards an immunosuppressive, M2-like phenotype. We undertook ex-vivo and in-vitro experiments to probe TAM- T cell interactions upon E2 treatment. Using this approach, it was determined that E2 did not directly affect T-cell activity but resulted in the suppression of T-cell activity secondary to its actions on TAMs. TAMs from E2-treated tumors suppressed T-cell proliferation, activation, and secretion of cytotoxic granules, and genetic ablation of ER in TAMs reversed the ability of TAMs to suppress T-cell activity upon E2 treatment. While this work discovered the clinical utility of targeting ER signaling in TAMs in melanoma, these findings are currently being translated to other cancer types, especially those with a high intratumoral macrophage infiltration.

Overall, the work performed has unraveled the importance of estrogen signaling in the tumor immune microenvironment, opening new avenues for increasing anti-tumor immunity through the targeting of estrogen signaling in immune cells.

The estrogen receptor (ER/ESR1) is expressed in the majority of breast and gynecological cancers. As such, drugs that inhibit ER signaling are the cornerstone of pharmacotherapy for these malignancies. Treatment strategies include the Selective Estrogen Receptor Modulator (SERM) tamoxifen, which acts as a competitive antagonist, and aromatase inhibitors (AIs) drugs that inhibit the enzyme responsible for the production of 17- estradiol (E2), the most biologically important estrogen. However, the clinical utility of these treatment strategies are limited by the development of de novo and acquired resistance. The mechanisms underlying resistance to these endocrine therapies are varied and complex include activating genomic alterations in ER (amplification, translocations, and mutations), cell cycle dysregulation and activation of alternative growth factor signaling pathways. Interestingly, it has been observed that ER signaling remains engaged and targetable in the majority of these tumors at all stages of disease. As such, the selective estrogen receptor downregulator (SERD) fulvestrant, which is both a competitive antagonist and downregulator of ER, is often used to treat tumors progressing on AIs or tamoxifen. However, the unfavorable pharmacokinetic properties of this drug have largely limited its use as a monotherapy creating a need for additional ER-modulators.

The field has put much effort into developing orally bioavailable, next-generation SERDs to replace fulvestrant in advanced breast cancer. However, many early efforts to optimize compounds for their degradation activity has not yielded clinically useful drugs. Notwithstanding issues related to drug exposure which may have impacted efficacy there is significant data to suggest that “antagonist activity” is the primary driver of SERD efficacy. To address the need to replace or optimize fulvestrant therapy for advanced breast cancer we undertook both unbiased and biased approaches to define new therapeutic strategies that target ER.

In the first set of studies, we investigated the impact of mutations in ESR1, which occur in metastatic lesions, may have on receptor pharmacology. Specifically, activating point mutations within the ligand binding domain (LBD) of ESR1 have presented as a mechanism of acquired resistance to AIs in metastatic breast cancer; as well as in both de novo and acquired resistance in primary gynecological cancers. Interestingly, these mutations are also resistant/partially resistant to many clinically relevant SERMs and SERDs, including tamoxifen and fulvestrant. Therefore, we undertook a study to elucidate the molecular mechanism(s) underlying ESR1 mutant pharmacology in relevant models of breast cancer. These studies revealed, unexpectedly, that the response of ESR1 mutations to various ligands was dictated primarily by the relative coexpression of ERWT in cells. Specifically, altered pharmacology was only evident in cells in which the mutants were overexpressed relative to ligand-activated ERWT. Importantly, while undertaking an unbiased approach to evaluate all clinically relevant antagonists for activity on the ESR1 mutants, we made the serendipitous discovery that the antagonist activity of the SERM lasofoxifene was not impacted by mutant status. This finding has led to its clinical evaluation as a treatment for patients with advanced ER-positive breast cancer whose tumors harbor ESR1 mutations, with additional studies in patients with gynecological cancer patients likely to be undertaken in the near future.

In addition to the unbiased approach outlined above we also approached the problem of resistance taking a candidate approach to evaluate structurally distinct SERDs, as monotherapy and in combination with CDK 4/6 inhibition, in relevant models of advanced breast cancer. G1T48 is a novel orally bioavailable, non-steroidal small molecule antagonist that we demonstrated both in vitro and in vivo has the potential to be an efficacious oral antineoplastic agent in ER positive breast cancer. While G1T48 can effectively suppress ER activity in multiple models of endocrine therapy resistance, this compound still displayed partial resistance to the ERmuts.

Together, our data supports the hypothesis that novel compounds targeting ER should be optimized based on antagonist potential and not on degradative activity per se. As such, the results of these studies will inform the development of next-generation therapeutics for endocrine therapy resistant cancers, especially those harboring ESR1 mutations.

During the immune response, helper T cells must proliferate and upregulate key metabolic programs including glucose and glutamine uptake. Metabolic reprogramming is imperative for appropriate T cell responses, as inhibition of glucose or glutamine uptake hinders T cell effector responses. Glutamine and glutaminolysis use in cancer cells has partially been explored. However, the role of glutamine and its downstream metabolites is incomplete and unclear in T cells. The first step of glutamine metabolism is conversion to glutamate via the hydrolase enzyme glutaminase (GLS). To target glutaminolysis, two different methods were employed: 1) genetic knockout of GLS using a CRE-recombinase system specific for CD4/CD8 T cells, and 2) pharmacological inhibition of GLS via the potent and specific small molecular CB839. These two models of glutaminase insufficiency were used as a tool to target glutamine metabolism during T cell activation and differentiation both in vitro and in vivo.

GLS-deficient T cells had decreased activation at early time points compared to control. Over several days, these GLS-deficient T cells differentiated preferentially to Th1-like effector cells. This was reliant on increased glucose carbons incorporating into Tri-Carboxylic Acid (TCA) metabolites. This increased effector response in vitro occurred in both CD4+ T helper cells and CD8+ cells (Cytotoxic lymphocytes, or CTLs). Differentiation of CD4+ T cells to Th1 or Th17 subsets showed decreased Th17 differentiation and cytokine production, while Th1 effector responses were increased. This increased Th1 function was dependent on IL-2 signaling and mTORC1, as reducing IL-2 or inhibiting mTORC1 with rapamycin prevented GLS inhibition-induced Th1 effector function. Th17 cells, meanwhile, were inhibited by changes in reactive oxygen species, and recovery of Th17 function was achieved with n-acetylcysteine treatment.

T cells lacking GLS were unable to induce inflammation in a mouse model of Graft vs Host disease, an inflammatory bowel disease model, or in an airway inflammatory model. Importantly, Chimeric Antigen Receptor (CAR) T cells made from GLS knockout cells were unable to maintain B cell aplasia in recipient mice. Contrary to this, temporary inhibition of GLS via small-molecule inhibition increased B cell killing in vitro and enhanced T cell persistence in both the B cell aplasia and in a vaccinia virus recall response. These results indicate a balance, where permanent deficiency of GLS is detrimental to T cell responses, but acute inhibition can actually promote T effector responses and survival. Overall, this work aims to understand how perturbations in glutamine metabolism in T cells affects differentiation and function and the role of glutaminolysis and improve therapies for inflammatory disease and cancer.

Prostate and breast cancers are major health concerns, being amongst the most common forms of cancers in both men and women. The majority of prostate and breast cancers are driven by the hormone receptors androgen receptor (AR) and estrogen receptor (ESR1), respectively, and as such, endocrine therapies targeting the actions of these receptors has been a cornerstone of treatment for these patients. While these endocrine therapies are generally initially efficacious, resistance inevitably emerges. Resistance can emerge through various mechanisms, such as amplification of the receptor, generation of activating point mutations, alternative splicing of the receptor resulting in constitutively active forms of the receptor, and activating cross-talk from growth factor signaling pathways. A salient feature of these diseases is that the nuclear receptor (AR or ER) often remains engaged upon the emergence of resistance, and thus targeting of the receptor still provides therapeutic benefit. Therefore, much work in these fields has been performed to design better forms of endocrine therapy to help patients upon tumor progression. As cells are altering their signaling to deal with these pressures, this thesis work investigated the global genomic changes which arise in prostate and breast cancer cells after endocrine therapy to understand the effects of utilizing different forms of endocrine therapy, and whether these alterations in the cells induce novel vulnerabilities which can be therapeutically exploited. In the first set of studies, the differences between utilizing a competitive antagonist (enzalutamide-Enz) vs an AR degrader (AR-targeting proteolysis targeting chimera-PROTAC) were evaluated in prostate cancer. PROTACs are a new form of therapy for prostate cancer which have encouraging results in early clinical trials, so we wanted to better understand the genomic architecture and gene expression landscape after this new treatment modality compared to the current standard of care with an aim to use this knowledge to understand endocrine therapy resistance and identify therapeutically targetable pathways emerging from treatment. A factor agnostic approach was taken utilizing ATACseq and RNAseq to compare the genomic landscape after Enz or PROTAC treatment. It was found that the different AR inhibitors create distinct genomic landscapes which appear to be driven by unique sets of transcription factors. Further, it was discovered that AR inhibition, especially through degradation creates a novel liability which can be therapeutically exploited. AR was found to mediate these effects through regulating expression of a key transcription factor, and we propose a model in which the two proteins interact to regulate this axis. As AR is expressed in many other malignancies, it is feasible this strategy of degrading AR to induce this therapeutic vulnerability could have efficacy beyond prostate cancer. In the second set of studies, we investigated the genomic changes which are manifest after the emergence of endocrine therapy resistance in breast cancer and identified a novel signaling pathway that, when targeted, impairs tumor progression. Utilizing, DNAse hypersensitivity analysis, ChIP-seq, and RNAseq, it was found that GRHL2 cooperates with FOXA1 to drive a novel cistrome in endocrine therapy resistant breast cancer cells. The protein LYPD3 was found to be a downstream effector of GRHL2 and targeting LYPD3, or its ligand AGR2, with monoclonal antibodies significantly impaired primary tumor growth. Further studies into the functional role of LYPD3 were then undertaken, and it was discovered that LYPD3 knockdown significantly alters metastatic outgrowth of breast cancer cells in the lung. Investigation into the signaling of LYPD3 revealed a novel function of this protein. This work and future mechanistic studies will elucidate the signaling of LYPD3, and as LYPD3 is expressed in numerous subtypes of advanced cancers, understanding its signaling could provide a new biomarker for cancers which would be amenable to the targeted therapies identified in these studies in combination with LYPD3 targeted therapies.

The majority of breast tumors express the estrogen receptor (ER), and more than half of these cancers also express the progesterone receptor (PR). While the actions of ER on breast cancer pathogenesis are well understood, those of PR are still unclear. The Women's Health Initiative trial in 2002 brought into focus the alarming result that women receiving both estrogen and progestins as hormone replacement therapy are at greater risk for breast cancer than women receiving estrogen alone. Thus, there is considerable interest in defining the mechanisms that underlie the pharmacological actions of progestins in the normal and malignant breast.

Progestins facilitate cell cycle progression through multiple mechanisms, one of which is the induction of phosphorylation of the tumor suppressor retinoblastoma (Rb) protein. Stimulation by growth factors induces the transcription of Cyclin D1 which in turn activates the cyclin dependent kinases (CDKs). The Cyclin D1- Cdk4/6 complex phosphorylates the Rb protein, leading to the release of E2F1, which then binds and activates other target genes, leading to G1-S transition of the cell cycle. Given the reported action of PR to activate MAPK signaling, we initially thought that the progestin-induced Rb phosphorylation was mediated by this pathway. However, we turned to an alternate hypothesis based on our data using MEK inhibitors demonstrating that this was not the case.

Given the primacy of Cyclin D1 in cell cycle control, we then turned our attention to defining the mechanism by which Cyclin D1 expression is regulated by PR. Interestingly, it was determined that progestin mediated up- regulation of Cyclin D1 is rapid, peaking at 6hrs post hormone addition followed by a decrease in expression reaching a nadir at 18hrs. Unexpectedly, we found that contrary to what has been published before, the induction of Cyclin D1 mRNA expression was a primary transcriptional event and we have demonstrated the specific interaction of PR with PREs (progesterone response elements) located on this gene. We have further determined that the half-life of Cyclin D1 mRNA is decreased significantly by progestin addition explaining how the levels of this mRNA following the addition of hormone are quickly attenuated. Thus, when taken together, our data suggest that progestins exert both positive and negative effects on Cyclin D1 mRNA, the uncoupling of which is likely to impact the pathogenesis of breast cancer

The observation that PR reduces the Cyclin D1 mRNA stability led us to investigate the effects of PR on RNA binding proteins, especially those which are involved in RNA stability. We discovered that PR induces the expression of several RNA binding proteins. Although the work to determine the effects of these RNA binding proteins on CyclinD1 mRNA stability is still ongoing, we have discovered a role for one of the PR-induced RNA binding proteins tristetraprolin (TTP), in the suppression of the inflammation pathway in breast cancer. We found that while TTP was not required for the PR-mediated decrease in Cyclin D1 mRNA stability, overexpression of this tumor suppressive protein was able to inhibit IL-1β-mediated stimulation of inflammatory genes in our breast cancer model. Since it is established that the upregulation of the inflammatory pathway is oncogenic, we are currently exploring the intersection of PR and TTP-mediated signaling on the inflammation transcriptome in breast cancer.

Thus, collectively these data provide us with a better picture of the poorly understood actions of PR on breast cancer proliferation and tumorigenesis. We believe that further investigation of the studies developed in this thesis will lead to novel and better-targeted approaches to the use of PR as a therapeutic target in the clinic.

Proper balancing of the activities of metabolic pathways to meet the challenge of providing necessary products for biosynthetic and energy demands of the cell is a key requirement for maintaining cell viability and allowing for cell proliferation. Cell metabolism has been found to play a crucial role in numerous cell settings, including in the cells of the immune system, where a successful immune response requires rapid proliferation and successful clearance of dangerous pathogens followed by resolution of the immune response. Additionally, it is now well known that cell metabolism is markedly altered from normal cells in the setting of cancer, where tumor cells rapidly and persistently proliferate. In both settings, alterations to the metabolic profile of the cells play important roles in promoting cell proliferation and survival.

It has long been known that many types of tumor cells and actively proliferating immune cells adopt a metabolic phenotype of aerobic glycolysis, whereby the cell, even under normoxic conditions, imports large amounts of glucose and fluxes it through the glycolytic pathway and produces lactate. However, the metabolic programs utilized by various immune cell subsets have only recently begun to be explored in detail, and the metabolic features and pathways influencing cell metabolism in tumor cells in vivo have not been studied in detail. The work presented here examines the role of metabolism in regulating the function of an important subset of the immune system, the regulatory T cell (Treg) and the role and regulation of metabolism in the context of malignant T cell acute lymphoblastic leukemia (T-ALL). We show that Treg cells, in order to properly function to suppress auto-inflammatory disease, adopt a metabolic program that is characterized by oxidative metabolism and active suppression of anabolic signaling and metabolic pathways. We found that the transcription factor FoxP3, which is highly expressed in Treg cells, drives this phenotype. Perturbing the metabolic phenotype of Treg cells by enforcing increased glycolysis or driving proliferation and anabolic signaling through inflammatory signaling pathways results in a reduction in suppressive function of Tregs.

In our studies focused on the metabolism of T-ALL, we observed that while T-ALL cells use and require aerobic glycolysis, the glycolytic metabolism of T-ALL is restrained compared to that of an antigen activated T cell. The metabolism of T-ALL is instead balanced, with mitochondrial metabolism also being increased. We observed that the pro-anabolic growth mTORC1 signaling pathway was limited in primary T-ALL cells as a result of AMPK pathway activity. AMPK pathway signaling was elevated as a result of oncogene induced metabolic stress. AMPK played a key role in the regulation of T-ALL cell metabolism, as genetic deletion of AMPK in an in vivo murine model of T-ALL resulted in increased glycolysis and anabolic metabolism, yet paradoxically increased cell death and increased mouse survival time. AMPK acts to promote mitochondrial oxidative metabolism in T-ALL through the regulation of Complex I activity, and loss of AMPK reduced mitochondrial oxidative metabolism and resulted in increased metabolic stress. Confirming a role for mitochondrial metabolism in T-ALL, we observed that the direct pharmacological inhibition of Complex I also resulted in a rapid loss of T-ALL cell viability in vitro and in vivo. Taken together, this work establishes an important role for AMPK to both balance the metabolic pathways utilized by T-ALL to allow for cell proliferation and to also promote tumor cell viability by controlling metabolic stress.

Overall, this work demonstrates the importance of the proper coupling of metabolic pathway activity with the function needs of particular types of immune cells. We show that Treg cells, which mainly act to keep immune responses well regulated, adopt a metabolic program where glycolytic metabolism is actively repressed, while oxidative metabolism is promoted. In the setting of malignant T-ALL cells, metabolic activity is surprisingly balanced, with both glycolysis and mitochondrial oxidative metabolism being utilized. In both cases, altering the metabolic balance towards glycolytic metabolism results in negative outcomes for the cell, with decreased Treg functionality and increased metabolic stress in T-ALL. In both cases, this work has generated a new understanding of how metabolism couples to immune cell function, and may allow for selective targeting of immune cell subsets by the specific targeting of metabolic pathways.

The estrogen-related receptor alpha (ERRα) is an orphan nuclear receptor (NR) with no natural ligand identified. Recent studies report that ERRα expression and activity correlate with poor prognosis in breast cancer. It is also suggested that ERRα is involved in tumor growth and progression, thus this receptor may be a therapeutic target in the treatment of breast cancer. However, the specific role of ERRα in breast cancer is not fully understood. Similar to other nuclear receptors, ERR has been suggested to regulate target gene transcription through both classical (direct DNA binding) and non-canonical (tethering mechanisms) to effect various aspects of tumor pathogenesis, such as angiogenesis, regulation of hypoxic response, tumor growth, and migration. Thus, the objective of this dissertation research is to explore the roles of ERRα in breast cancer by (a) identifying novel ERRα target genes important for tumor pathogenesis, (b) characterizing the molecular mechanism of non-canonical actions of ERRα-mediated gene transcription, and (c) examining the structure basis of ERRα antagonism for future pharmaceutical exploitation. First, we identified an ERRα target gene, ECM1, which is relevant to breast cancer angiogenesis. The role of ECM1 in angiogenesis was confirmed by endothelial tube formation assay. We further showed that knocking down ECM1 has a dramatic inhibitory effect on tumor xenograft growth. This result, for the first time, directly demonstrates the role of ECM1 in tumor environment and further sheds light on the significance of ERR&alpha-regulated genes in tumors angiogenesis. Next, we explored the molecular mechanism of ERRα non-canonical pathways using transcriptional reporter assay and ERRα DNA-binding domain (DBD) mutants. We discovered that the expression of carbonic anhydrase 9 (CA9), a target gene of one of the ERRα tethering partner hypoxia inducible factor-1 (HIF-1), does not require direct binding of ERRα to DNA but its DBD is indispensible. These results reflect on the importance of ERRα DBD even in the non-canonical signaling of ERRα, which brings challenges to dissecting ERRα canonical/non-canonical pathways in the future. Finally, to determine the molecular mechanisms underlying ERRα antagonism, we probed the conformations of ERRα upon antagonist treatments. M13 phage display was used to screen for ERRα-interacting peptides. We identified peptides that interact with ERRα in the activation function 2 (AF2) domain, some of which are able to distinguish the binding of different classes of ERRα antagonists. Cumulatively, these studies have explored the biological functions of ERRα and the molecular basis ERRα-mediated signaling pathways.

Medulloblastoma (MB) is a highly malignant brain tumor that occurs primarily in children. Although surgery, radiation and high-dose chemotherapy have led to increased survival, many MB patients still die from their disease, and patients who survive suffer severe long-term side effects as a consequence of treatment. Thus, more effective and less toxic therapies for MB are critically important. Identifying new treatments will require an understanding of early stages of tumor development - the cell types from which the tumors arise and the signals that regulate their growth - as well as identification of pathways that are critical for the growth and maintenance of established tumors.

In these studies, we first explore the role of WNT signaling in cerebellar progenitors and their potential to serve as cells of origin for WNT-driven tumors. The WNT pathway plays multiple roles in neural development, is crucial for establishment of the embryonic cerebellum, and is highly expressed in a subset of MBs. However, the cell types within the cerebellum that are responsive to WNT signaling remain unknown. We show that expression of activated β-catenin promotes proliferation of cerebellar neural stem cells (NSCs) but not granule neuron precursors (GNPs). Although β-catenin expressing NSCs proliferate in vivo they do not undergo prolonged expansion or neoplastic growth; rather, WNT signaling markedly interferes with their capacity for self-renewal and differentiation. At a molecular level, mutant NSCs exhibit increased expression of c-Myc, which might account for their transient proliferation, but also express high levels of bone morphogenetic proteins and the cyclin-dependent kinase inhibitor p21, which might contribute to their altered self-renewal and differentiation. These studies suggest that the WNT pathway is a potent regulator of cerebellar stem cell growth and differentiation and that cooperating "second hits" may be required for neoplastic transformation.

In addition to understanding early stages of transformation, identifying vulnerabilities of established tumors will be critical for development of targeted therapies. Our studies in Chapter 3 are focused on the role of Survivin in SHH-driven MB and utility of survivin inhibition as a therapeutic approach for MB. Survivin is an inhibitor of apoptosis protein (IAP) that regulates cell cycle progression and resistance to apoptosis, is frequently expressed in human MB, and when expressed at high levels predicts poor clinical outcome. Here we show that Survivin is overexpressed in tumors from patched (Ptch) mutant mice, a model of Sonic hedgehog (SHH)-driven MB. Using genetic and pharmacological approaches, we demonstrate that inhibition of Survivin impairs proliferation and survival of both murine and human MB cells. Although Survivin antagonists do not cross the blood-brain barrier, they are capable of impeding growth of MB cells in flank allografts. These studies highlight the importance of Survivin in SHH-driven MB, and suggest that it may represent a novel therapeutic target in patients with this disease.

The progesterone receptor (PR) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors. The steroid hormone progesterone binds to PR and induces a conformational change that enables the receptor to bind DNA, recruit cofactors, and directly regulate the transcription of target genes. In addition, extra-nuclear PR can indirectly regulate gene expression by rapidly activating other signaling pathways such as Src/MAPK. Although the direct and indirect functions of PR have been well studied in isolation, it is important to understand the molecular mechanisms by which these pathways can cross talk and integrate to ultimately impact gene expression.

Towards this end, we initiated studies to assess the overall impact of MAPK inhibition on PR transcriptional activity in T47D breast cancer cells treated with the synthetic progestin R5020. During the course of microarray and biochemical analyses that were undertaken to address this issue, we discovered a subset of PR target genes that are enriched for E2F binding sites. Subsequently, we determined that PR-B is a component of several distinct pathways that function both directly and indirectly to positively up-regulate E2F1 expression in T47D breast cancer cells. Firstly, PR directly regulates E2F1 transcription by binding to proximal and distal enhancer sites located near E2F1. Secondly, progestin induces the hyperphosphorylation of Rb, which results in increased recruitment of E2F1 to its own promoter, thereby activating a positive feedback loop that further amplifies its transcription. Finally, PR induces expression of Krüppel-like factor 15 (KLF15) and potentially other Sp/KLF family members, which can bind to GC-rich DNA within the E2F1 promoter and further activate transcription. Together, these results suggest a paradigm for multimodal PR gene regulation that entails cooperation between direct and indirect pathways of PR signaling to achieve the desired downstream transcriptional cascade.

In the breast and other tissues of the female reproductive system, progesterone plays an important role in normal development and function. Therefore, synthetic PR modulators (PRMs) are widely used to manipulate the downstream biology of PR for purposes including contraception and hormone replacement therapy (HRT). However, progestins and PR have also been implicated in disease pathologies such as breast cancer. While the molecular mechanisms by which PR regulates breast tumor growth have not been fully elucidated, recent studies highlight the fact that progestins may have a dose-dependent role in breast cancer progression. Consequently, we undertook studies to identify and characterize any differential effects of low-dose versus high-dose progestins on the downstream activities of PR. Specifically, we found that treatment of breast cancer cells with low-dose progestins can induce maximal transcriptional activation of a subset of PR target genes, including the cell cycle regulators cyclin D1 and E2F1. Furthermore, low-dose and high-dose progestins have differential effects on the phosphorylation of PR and subsequent receptor turnover. Cumulatively, these findings underscore the importance of establishing the effects of a wide range of progestin concentrations on target gene expression and other PR actions, so that we are able to accurately predict the potential consequences of PRMs on downstream PR signaling pathways and biology.

Angiogenesis, the production of new vessels from pre-existing vasculature, is a complex biological process that is dependent on a series of regulated events, including endothelial cell (EC) proliferation, migration, survival, and capillary morphogenesis (tube formation). These events are required for angiogenesis to occur properly and the steps are regulated by a variety of vascular growth factors and their receptors. Tie2, an endothelial receptor tyrosine kinase (RTK), is required for embryonic and postnatal angiogenesis. Studies have demonstrated that Tie2 is proteolytically cleaved, producing a 75 kDa soluble receptor fragment (sTie2). However, the mechanisms and function of sTie2 have not been elucidated. Here, we investigated signaling pathways and effector molecule(s) responsible for Tie2 cleavage. Additionally, we investigated the role of other growth factors and conditions on the degree of Tie2 cleavage. Finally, we examined sTie2 levels in peripheral artery disease, a human model of ischemic disease. We demonstrated that Tie2 cleavage is VEGF- and PI3K/Akt-dependent and sTie2 can bind Ang1 and Ang2 and prevent ligand-mediated Tie2 activation and downstream cellular responses. Also, ADAM15 cleaves Tie2 in a hypoxia-dependent manner and this response was also observed to be VEGF-mediated. With respect to peripheral artery disease, sTie2 levels were only significantly elevated in the most angiogenically compromised group (critical limb ischemia) of patients. These data shed light on the mechanism and function of Tie2 cleavage and suggest a role for sTie2 in mediating the angiogenic process.

The androgen receptor (AR) signaling axis is a well-established therapeutic target in prostate cancer, due to its central role in tumor maintenance and progression. Although patients respond initially to androgen deprivation therapies and AR antagonists, they invariably progress to a castration-resistant state. Consequently, there is an unmet need for agents that target the AR signaling axis in a unique manner.

Histone deacetylase (HDAC) inhibitors repress AR signaling and prostate cancer growth in cellular and xenograft models. However, HDAC inhibitors also induce epithelial to mesenchymal (EMT) and neuroendocrine differentiation, both of which are associated with prostate cancer progression and aggressiveness. Given that 18 different HDAC isoforms have been identified in humans, and non-selective or Class I (HDAC1, 2, 3, and 8) HDAC inhibitors have been used in most of these studies, the relative contribution of individual HDAC isoforms to AR transcriptional activity and prostate cancer pathophysiology remains to be elucidated. The overarching goals of this study were to (1) determine the role of individual Class I HDACs in AR transcriptional activity and prostate cancer growth, (2) identify selective HDAC inhibitors that have reduced adverse profiles to the treatment of prostate cancer, and (3) identify potential HDAC-interacting proteins that regulate AR target gene transcription and prostate cancer growth.

Using genetic knockdown studies and pharmacological inhibitors with isoform selectivity, we identified that HDAC3 was required for AR transcriptional activity and proliferation in cellular models of androgen-sensitive and castration-resistant prostate cancer (CRPC). Additionally, we found that RGFP966, an HDAC3-selective inhibitor, attenuated the growth of a xenograft model of CRPC. Furthermore, non-selective HDAC inhibitors induced EMT and neuroendocrine markers in prostate cancer cells, but RGFP966 treatment did not. These studies provide rationale for selective inhibition of HDAC3 for the treatment of CRPC, and could provide an explanation for the lack of success using non-selective HDAC inhibitors in clinical trials for prostate cancer.

We also assessed the role of REV-ERB alpha, an HDAC3-interacting protein, in the regulation of AR transcriptional activity and prostate cancer growth. Using siRNA knockdown studies, REV-ERB inhibitors, and overexpression studies, we concluded that REV-ERB alpha; was required for AR target gene induction and prostate cancer growth, including models of CRPC. These studies also provide rational for targeting REV-ERB alpha; for the treatment of CRPC.

Taken together, these studies identify two novel targets in the HDAC signaling axis for the treatment of prostate cancer: HDAC3 and REV-ERB alpha. Our studies provide greater insight into AR transcriptional regulation and prostate cancer pathophysiology.